Anchorage dependant cells are typically cultured in the presence of surfaces or media containing animal-derived components, such as feeder layers, serum, collagen, fibronectin, vitronectin, Matrigel™, and the like, to facilitate anchoring of the cells to a surface.
One advantage of many animal-derived biological coatings is their ability to be prepared using simple protocols, which can be practiced without special and cumbersome equipment. For example, coatings made from cell adhesion proteins are performed by dissolving the proteins in water at an appropriate concentration and pH, dispensing the appropriate volume onto the surface of the article to be coated, incubating for an appropriate time and temperature, and rinsing to wash off the unbound materials. Typically no chemical crosslinking step is required, which avoids the use of specific chemical or physical processes for stable immobilization of the cell adhesion proteins on the article surface.
However, animal-derived additions to the culture environment expose cells to potentially harmful viruses or other infectious agents, which could compromise general culture and maintenance of the cells and could be transferred to patients if the cells or products of the cells are to be used for therapeutic purposes. In addition, such biological products are vulnerable to batch variation, immune response and limited shelf-life.
Therefore, methods of producing synthetic cell culture surfaces that are capable of supporting cells in chemically defined or serum-free media, are desirable. This is particularly true for cells that may be used in patients for therapeutic purposes, such as pluripotent stem cells, which have the ability to differentiate into any of the three germ layers, giving rise to any adult cell type.
To overcome the risk of contamination and batch variation, recombinant proteins having cell-adhesive properties have been proposed. However, such techniques are often complex, require extensive purification, and are expensive. Others have proposed in situ formation of swellable polymers from monomers to which cell adhesive polypeptides may be grafted. However, such techniques require careful control of casting of monomers and solvent evaporation to produce homogenous surfaces, complex equipment for UV curing, and complex chemistry for polypeptide grafting.
Another way to solve the issues encountered with biological coatings includes coating water-insoluble polymers and associated cell adhesive polypeptides or ligands on the surfaces of cell culture articles. The polymers and associated cell adhesive polypeptides are water-insoluble so that they do not release or dissolve in the presence of an aqueous cell culture medium. However, as the polymer-polypeptide/ligand polymers are water-insoluble, they cannot be used for coating from aqueous solution as done usually with biological attachment factors. In addition, such processes tend to suffer from inhomogeneous coating without careful control of dispensing and solvent evaporation and may not be practicable with various formats such as large vessels or small wells from multiwell plates due to variable evaporation rates occurring within such different formats.
There is still a need for a simple coating process using a synthetic polymer to prevent potential xenogeneic contamination and batch to batch variability but that can be performed simply as usually done with animal derived biological attachment factors.